Role of M2 muscarinic receptors in airway smooth muscle contraction.

Airway smooth muscle expresses both M2 and M3 muscarinic receptors with the majority of the receptors of the M2 subtype. Activation of M3 receptors, which couple to Gq, initiates contraction of airway smooth muscle while activation of M2 receptors, which couple to Gi, inhibits beta-adrenergic mediated relaxation. Increased sensitivity to intracellular Ca2+ is an important mechanism for agonist-induced contraction of airway smooth muscle but the signal transduction pathways involved are uncertain. We studied Ca2+ sensitization by acetylcholine (ACh) and endothelin-1 (ET-1) in porcine tracheal smooth muscle by measuring contractions at constant [Ca2+] in strips permeabilized with Staphylococcal alpha-toxin. Both ACh and ET-1 contracted airway smooth muscle at constant [Ca2+]. Pretreatment with pertussis toxin for 18-20 hours reduced ACh contractions, but had no effect on those of ET-1 or GTPgammaS. We conclude that the M2 muscarinic receptor contributes to airway smooth muscle contraction at constant [Ca2+] via the heterotrimeric G-protein Gi.

Hypocapnia-induced contraction of porcine airway smooth muscle.

Hypocapnia constricts peripheral airways in vivo. This study investigated the role of airway smooth muscle in this phenomenon and the mechanism of hypocapnia-induced contraction in vitro. Isometric tension, intracellular pH (pHi) and intracellular free calcium concentration ([Ca2+]i) were measured in porcine airway smooth muscles suspended in organ baths in the presence of 5% or 0% CO2. In tracheal strips precontracted with carbachol, hypocapnic challenge (0% CO2) produced increases in tension, pHi, and [Ca2+]i. In bronchial rings or tracheal strips precontracted with carbachol, nifedipine administered between consecutive contractions attenuated responses to hypocapnia (75+/-11% above carbachol-precontracted tension before nifedipine versus 39+/-9% after nifedipine, n=7 bronchial rings, p<0.05). Neither indomethacin (5 microM), nordihydroguaiaretic acid (10 microM) nor phenidone (10 microM) significantly altered responses. These data suggest that enhanced Ca2+ influx through voltage-dependent Ca2+ channels of airway smooth muscle cells is important in airway responses to hypocapnia.

Role of G proteins in agonist-induced Ca2+ sensitization of tracheal smooth muscle.

Increased sensitivity to intracellular Ca2+ concentration ([Ca2+]) is an important mechanism for agonist-induced contraction of airway smooth muscle, but the signal transduction pathways involved are uncertain. We studied Ca2+ sensitization with acetylcholine (ACh) and endothelin (ET)-1 in porcine tracheal smooth muscle by measuring contractions at a constant [Ca2+] in strips permeabilized with alpha-toxin or beta-escin. The peptide inhibitor G protein antagonist 2A (GP Ant-2A), which has selectivity for Gq over Gi, inhibited contractile responses to ET-1, ACh, and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), but the proportional inhibition of ACh responses was less than that of ET-1. Pretreatment with pertussis toxin reduced ACh contractions but had no effect on those of ET-1 or GTPgammaS. Clostridium botulinum C3 exoenzyme, which inactivates Rho family monomeric G proteins, caused similar reductions in contractile responses to ACh, ET-1, and GTPgammaS. Farnesyltransferase inhibition, which inhibits Ras G proteins, reduced responses to ET-1. We conclude that the heterotrimeric G proteins Gq and Gi both contribute to Ca2+ sensitization by ACh, whereas ET-1 responses involve Gq but not Gi. Both Gq and Gi pathways likely involve Rho family small G proteins. A Ras-mediated pathway also contributes to Ca2+ sensitization by ET-1 in airway smooth muscle.

Inhibition of voltage-dependent Ca2+ channels of porcine tracheal smooth muscle by the novel Ca2+ channel antagonist RWJ-22108.

1. We compared electrophysiological effects of the bronchoselective Ca2+ channel antagonist RWJ-22108 on voltage-dependent Ca2+ channels (VDCs) of porcine tracheal smooth muscle cells to the effects of nicardipine and verapamil. 2. Each of the three Ca2+ channel antagonists tested inhibited inward Ca2+ currents (ICa) measured by whole-cell patch clamp techniques. Inhibition was dose-dependent with approximately 50% inhibition of peak ICa at +20 mV obtained with 3 x 10(-6) M RWJ-22108, 3 x 10(-7) M nicardipine, or 10(-5) M verapamil. 3. Both RWJ-22108 (3 x 10(-6) M) and nicardipine (3 x 10(-7) M) shifted the voltage dependence of steady-state inactivation to more negative potentials; however, the change in the potential of half-maximal inactivation induced by RWJ-22108 (-18 mV) was significantly greater than that induced by nicardipine (-12 mV). Verapamil did not alter the voltage dependence of inactivation. 4. We conclude that inhibition of VDCs by RWJ-22108 is qualitatively similar to that by nicardipine but with a greater stabilizing effect on the inactivated channel state.

Role of calcium channel blockade in relaxation of tracheal smooth muscle by extracellular Mg2+.

Magnesium ion (Mg2+) is a bronchodilator, but little is known about its mechanism of action on airways. We hypothesized that Mg2+ inhibits voltage-dependent Ca2+ channels of airway smooth muscle. Smooth muscle cells were freshly dispersed from pig trachea with collagenase. Depolarization-induced inward Ca2+ currents, measured in whole cell patch-clamp experiments, were inhibited by nifedipine and stimulated by BAY K 8644. Increasing bath Mg2+ from 1 to 21 mM caused a reversible approximately 30% inhibition of current and a positive shift of the peak current-voltage relationship. Voltage-dependent steady-state inactivation had a half-maximal potential (Vh) of -12 mV and a Boltzmann slope factor (k) of 6.0 mV. High Mg2+ caused a positive shift in Vh without affecting k, whereas nifedipine caused a negative shift in Vh and increased k. The inhibition of voltage-dependent Ca2+ channel currents by Mg2+ was quantitatively similar to Mg(2+)-induced relaxation of KCl-contracted tracheal smooth muscle strips. We conclude that inhibition of Ca2+ influx through dihydropyridine-sensitive, voltage-dependent channels by Mg2+ accounts for much of its relaxant action on airway smooth muscle.

Ozone-induced vagal reflex modulates airways reactivity in rabbits.

We examined the effects of ozone (O3) on central and peripheral airway reactivity and tracheal transepithelial potential difference (PD) in New Zealand white rabbits. Rabbits were exposed for 7 h to either room temperature-humidified filtered air (n = 7) or 0.2 ppm O3 in humidified room air (n = 5). Tracheal PD was recorded 3 h after exposure. Whole lung resistance (RL) and reactivity were partitioned into their central (RC) and peripheral (RP) components using a retrograde catheter and forced oscillation. Changes in RL, RC, and RP in response to NaCl (0.9%) and ACh (100 mM) aerosol challenges were measured before and after vagotomy. Exposure to O3 decreased tracheal PD from -29 +/- 0.6 mV in air-exposed rabbits to -15 +/- 2 mV in O3-exposed rabbits (p < or = 0.0001). Exposure to O3 did not alter RL, RC, or RP. However, the ACh-induced increase in RL in O3-exposed rabbits (140%) was twice that recorded in the air-exposed group (p < or = 0.01). While changes in RP dominated the whole lung response to ACh in air-exposed rabbits, changes in RC were most prominent in the O3-exposed group. Bilateral vagotomy did not alter airway reactivity in control rabbits but did enhance peripheral lung reactivity in O3-exposed rabbits. We conclude that exposure to 0.2 ppm O3 for 7 h affects tracheal epithelial function in rabbits and increases central airway reactivity via vagal mechanisms without altering baseline RL, RC, or RP.

Sodium nitroprusside stimulates Ca2+ -activated K+ channels in porcine tracheal smooth muscle cells.

To directly investigate the possible role of large-conductance Ca2+ -activated K+ (KCa) channels in nitro-vasodilator-induced relaxation of airway smooth muscle, we used cell-attached patch-clamp techniques to test the effects of sodium nitroprusside (SNP) on KCa channels in freshly dispersed porcine tracheal smooth muscle cells. Channel open-state probability (nPo) increased approximately 13-fold with exposure to 10(-5) M SNP, and this was partially reversed by addition of the guanylate cyclase inhibitors methylene blue (3 X 10(-4) M) or LY-83583 (5 X 10(-5) M). Pretreatment with the guanosine 3',5' -cyclic monophosphate (cGMP)-dependent protein kinase (G kinase) inhibitor Rp-8-(p-chlorophenylthio) cGMP-phosphorothioate (2 X 10(-5) M) prevented activation of KCa channels by SNP. We also tested the ability of G kinase to directly activate KCa channels in inside-out patches. G kinase (2.5 U/microliter) with ATP (0.5 mM) and cGMP (0.1 mM), but not ATP and cGMP alone, increased nPo approximately 23-fold. We conclude that SNP activates KCa channels in airway smooth muscle via guanylate cyclase and G kinase. Phosphorylation of the channel protein by G kinase may account for this response. Consequent membrane hyperpolarization and inhibition of Ca2+ entry through voltage-dependent channels may contribute to SNP-induced relaxation of airway smooth muscle.

MgSO4 relaxes porcine airway smooth muscle by reducing Ca2+ entry.

Magnesium sulfate (MgSO4) is used clinically, but its mechanism of action is unknown. To determine whether MgSO4 relaxes airway smooth muscle and to investigate the pathways involved, we compared effects of MgSO4 in porcine tracheal and bronchial muscles contracted with either carbachol or KCl and measured the effects of MgSO4 on the concentration of intracellular free calcium ([Ca2+]i). Lungs were dissected after anesthesia and exsanguination. Tracheal strips and bronchial rings were suspended in tissue baths for measurement of isometric tension in the presence of different concentrations of MgSO4. In separate experiments, tracheal smooth muscle tension and [Ca2+]i were measured simultaneously, using the fluorescent dye fura 2. MgSO4 (1.2, 2.2, 9.2 mM) produced a concentration dependent rightward shift of contraction dose-response curves to KCl but not to carbachol. MgSO4 relaxed trachealis muscles precontracted with KCl or carbachol and simultaneously decreased [Ca2+]i. These findings indicate that MgSO4 directly relaxes airway smooth muscle and that the mechanism involves a decrease in [Ca2+]i. Because initiation and maintenance of contraction during KCl stimulation and maintenance of contraction during carbachol stimulation require Ca2+ entry through voltage-dependent calcium channels, MgSO4-induced relaxation may involve a decrease in Ca2+ entry via these channels.

Inhibitory effects of thiopental, ketamine, and propofol on voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells.

BACKGROUND: Intravenously administered anesthetics directly inhibit airway smooth muscle contraction. Because many anesthetic agents affect membrane ion channel function and sustained contraction of airway smooth muscle requires the influx of Ca2+ through voltage-dependent Ca2+ channels, it was hypothesized that intravenous anesthetics inhibit airway smooth muscle voltage-dependent Ca2+ channels. METHODS: Porcine tracheal smooth muscle cells were enzymatically dispersed and studied using whole-cell, patch-clamp techniques. The cells were exposed to thiopental (10(-7)-3 x 10(-4) M), ketamine (10(-6)-10(-3) M), or propofol (10(-7)-3 x 10(-4) M) while recording macroscopic voltage-activated Ca2+ currents (ICa). RESULTS: Each intravenous anesthetic tested significantly inhibited ICa in a dose-dependent manner with 3 x 10(-4) M thiopental, 10(-3) M ketamine, and 3 x 10(-4) M propofol each causing approximately 50% depression of peak ICa, but with no apparent shift in the voltage dependence of induced ICa. After pretreatment with the Ca2+ channel agonist Bay K 8644, thiopental, but not ketamine or propofol, shifted the maximum ICa to more positive potentials. All three anesthetics promoted the inactivated state of the channel at more negative potentials, but propofol was less effective than thiopental or ketamine in this regard. CONCLUSIONS: Three intravenous anesthetics evaluated in this study decreased the ICa of porcine tracheal smooth muscle cells but with subtle electrophysiologic differences. Hence, thiopental, ketamine, and propofol each inhibit L-type voltage-dependent Ca2+ channels of porcine tracheal smooth muscle cells but the molecular mechanisms involved may be agent specific. This inhibition may contribute to the airway smooth muscle relaxant effects of these agents observed in vitro at concentrations greater than those encountered clinically.

Cholinergic regulation of voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells.

To investigate cholinergic regulation of voltage-dependent Ca2+ channels (VDCs) in airway smooth muscle, we measured inward currents through VDCs in enzymatically dispersed porcine tracheal smooth muscle cells using conventional (10 mM Ca2+ as charge carrier) and nystatin-perforated (5 mM Ba2+ as charge carrier) whole cell patch clamp techniques. Carbachol (CCh) had significant and dose-dependent inhibitory effects on inward currents (12% with 10(-7) M and 42% with 10(-6) M) in perforated whole cell clamp experiments, but had no effect on currents in conventional whole cell experiments. CCh also shifted the steady-state inactivation curve to more negative potentials. Further experiments tested the hypothesis that CCh inhibits VDCs in part by the activation of protein kinase C (PKC). Phorbol 12,13-diacetate, an exogenous PKC activator, inhibited currents through VDCs. and calphostin C, a specific PKC inhibitor, antagonized the inhibitory effect of CCh. Furthermore, intracellular exposure to the activating PKC fragment 530-558, using a pipette perfusion technique, also inhibited currents through VDCs. We conclude that cholinergic receptor stimulation can inhibit inward Ca2+ currents through VDCs of porcine tracheal smooth muscle and that this effect may be mediated in part by activation of PKC.

Genetic control of susceptibility to ozone-induced changes in mouse tracheal electrophysiology.

Genetic factors influence the responses of humans and rodents to ozone (O3) inhalation. We previously demonstrated differential O3-induced decreases of tracheal potential (VT) in C57BL/6J (B6) and C3H/HeJ (C3) strain mice. To characterize the genetic basis of this strain-specific response, we measured VT in progeny of B6 and C3 strain mice and in six additional inbred strains of mice 6 h after O3 exposures (2 ppm x 3 h). First filial generation (F1) mice and second generation backcrosses with the resistant parent were uniformly resistant. The distribution of VT in second generation backcrosses with the susceptible parent resembled that of a population composed of resistant and susceptible mice in a 1:1 ratio. These data suggested simple autosomal recessive inheritance of susceptibility. However, overlapping distributions prevented statistical confirmation of that hypothesis. Strain screening revealed a susceptible phenotype in 129/J, A/J, B6, C3HeB/FeJ, and SJL/J and a resistant phenotype in AKR/J, C3, and CBA/J inbred mouse strains. Because this pattern of susceptibility to changes in VT differs from that of susceptibility to lung inflammation, the genetic factors that determine these two responses to acute O3 are not identical.

Intracellular pH regulates voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells.

Changes in CO2 or in pH modify airway smooth muscle contractility. To investigate the mechanisms involved, we compared K(+)-induced contractions in porcine bronchial rings exposed to different CO2 concentrations and directly measured the effects of changes in intracellular (pHi) or extracellular pH (pHo) on Ca2+ currents (ICa) through voltage-dependent Ca2+ channels (VDC) in porcine tracheal smooth muscle cells. Hypocapnia and hypercapnia caused leftward and rightward shifts, respectively, in the dose-response to K+ (P < 0.05) but did not change the maximum force obtained. Peak ICa (10 mM external Ca2+) elicited by depolarizing pulses from -80 mV was maximal [-265 +/- 12 pA (mean +/- SE), n = 19] at +10 mV. Intracellular acidification decreased the peak ICa at +10 mV from -261 +/- 20 pA to -177 +/- 12 pA (P < 0.05, n = 4), while intracellular alkalinization increased the peak ICa at +10 mV from -302 +/- 27 pA to -368 +/- 26 pA (P < 0.05, n = 4). Changes in pHo had little effect on ICa. There was no shift in the voltage-dependence of induced ICa with any change. We conclude that pHi, but not pHo, directly modulates the entry of Ca2+ into airway smooth muscle cells through VDC. This mechanism may contribute to regulation of airway tone by CO2.

Ozone-induced decrease of mouse tracheal potential is not secondary to cellular inflammation.

Acute exposure of C57BL/6J (B6) mice to ozone (O3) causes decreased in vivo tracheal electrical potential difference (PD). To investigate the role of inflammation in this response we measured O3 effects in B6 mice pretreated with indomethacin (5 mg/kg), colchicine (3 mg/kg), or cyclophosphamide (30 mg/kg x 7 days). Mice were exposed to 2 ppm O3 or air for 3 hr and allowed to recover for 0, 3, 6, 9, or 12 hr. Tracheal PD was measured under pentobarbital anesthesia using a capped agar bridge inserted into the upper trachea through a neck incision. After measurement of PD, bronchoalveolar lavage (BAL) was performed and total cells, polymorphonuclear leukocytes (PMNs), and protein were determined. As previously reported, O3 exposure decreased PD and increased BAL total cells, PMNs, and protein. O3-induced changes in PD and PMNs were maximal 6-9 hr after exposure. Indomethacin prevented the O3-induced change in PD but had no effect on BAL total cells or PMNs. Colchicine attenuated O3-induced increases in PMNs and cyclophosphamide decreased O3 effects on both BAL total cells and PMNs but neither drug affected the PD response. None of the drugs significantly altered O3-induced increases in BAL protein. The indomethacin sensitivity of O3-induced changes in PD may reflect a role of cyclooxygenase products in that response. However, drugs known to inhibit PMN function did not affect O3-induced changes in PD. We suggest that cellular inflammation is not required for the tracheal electrophysiological response to acute O3 exposure.

Volatile anesthetics inhibit voltage-dependent Ca2+ channels in porcine tracheal smooth muscle cells.

The relaxation of airway smooth muscle by volatile anesthetics is associated with a decreased concentration of intracellular free Ca2+. We hypothesized that inhibition of the entry of extracellular Ca2+ contributes to the relaxation. We therefore examined the effects of halothane, isoflurane, and sevoflurane on macroscopic voltage-activated Ca2+ currents (ICa) in porcine tracheal smooth muscle cells, using the whole cell patch-clamp technique. All three volatile anesthetics significantly inhibited ICa in a dose-dependent manner with no apparent shift in the voltage dependence of induced ICa. The order of inhibitory potencies for ICa was halothane > isoflurane > sevoflurane. When data were plotted as a function of the estimated anesthetic concentrations in the lipid phase, the potencies for inhibition of ICa by the three anesthetics were indistinguishable. We conclude that volatile anesthetics have an inhibitory effect on ICa of porcine tracheal smooth muscle cells at clinically relevant concentrations and that the inhibitory potencies of volatile anesthetics on ICa are closely related to their lipid-phase solubilities.

Role of intracellular pH in relaxation of porcine tracheal smooth muscle by respiratory gases.

Hypercapnia and hypoxia both relax airway smooth muscle, but the mechanisms responsible are poorly understood. Because hypercapnia and hypoxia can each decrease intracellular pH (pHi) and acidosis can inhibit Ca2+ channels, we hypothesized that decreased pHi mediates relaxation of trachealis muscle by each of these respiratory gases. To examine the relationship between pHi and tone, we measured isometric tension, bath pH, and fluorescence intensity (540 nm) in porcine tracheal smooth muscle strips loaded with 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein and excited alternately with 440- and 500-nm light. Strips equilibrated in Krebs-Henseleit solution bubbled with 95% O2-5% CO2 were contracted with carbachol and then relaxed with either 95% N2-5% CO2 or 93% O2-7% CO2. The ratio of fluorescence intensity at 500 nm to 440 nm was calibrated vs. pHi with use of nigericin. Baseline pHi was 7.19 +/- 0.03 (n = 13). Hypoxia decreased active tension by approximately 60% but did not change pHi. Hypercapnia induced decreases in tension that were associated with substantial decreases in pHi. Thus, decreased pHi does not mediate hypoxic relaxation, but the relaxation during physiologically relevant increases in CO2 concentration is associated with significant cellular acidification.

Inhibition of dihydropyridine-sensitive calcium entry in hypoxic relaxation of airway smooth muscle.

Hypoxia dilates airways in vivo and reduces active tension of airway smooth muscle in vitro. To determine whether hypoxia impairs Ca2+ entry through voltage-dependent channels (VDC), we tested the ability of dihydropyridines to modulate hypoxia-induced relaxation of KCl- and carbamyl choline (carbachol)-contracted porcine bronchi. Carbachol- or KCl-contracted bronchial rings were exposed to progressive hypoxia in the presence or absence of 1 microM BAY K 8644 (an L-type-channel agonist). In separate experiments, rings were contracted with carbachol or KCl, treated with nifedipine (a VDC antagonist), and finally exposed to hypoxia. BAY K 8644 prevented hypoxia-induced relaxation in KCl-contracted bronchi. Nifedipine (10(-5) M) totally relaxed KCl- contracted bronchi. Carbachol-contracted bronchi were only partially relaxed by nifedipine but were completely relaxed when the O2 concentration of the gas was reduced from 95 to 0%. These data indicate that hypoxia can reduce airway smooth muscle tone by limiting entry of Ca2+ through a dihydropyridine-sensitive pathway, but that other mechanisms also contribute to hypoxia-induced relaxation of carbachol-contracted bronchi.

Scaling of transepithelial potential difference in the mammalian trachea.

Tracheal epithelia of different mammalian species differ widely with regard to the relative rates of Na+ absorption and Cl- secretion. However, the short circuit current, a measure of total ion transport, appears to be consistently greater in large than in small mammals. Thus, we hypothesized that the in vivo tracheal electrical potential difference (PD) would vary among species as a function of body mass (M). To test this hypothesis we measured PD in ten mammalian species that ranged 1000-fold in mass. The results in mV (mean +/- SE, lumen negative) were: 11.4 +/- 1.0 in mice; 11.6 +/- 1.2 in gerbils; 12.9 +/- 1.4 in rats; 19.3 +/- 0.9 in guinea pigs; 27.2 +/- 2.2 in ferrets; 23.0 +/- 1.6 in cats; 27.0 +/- 0.6 in rabbits; 32.5 +/- 2.6 in dogs; 37.0 +/- 1.9 in sheep; and 49.0 +/- 3.3 in pigs. Log-log correlation analysis of mean PD (in mV) and M (in kg) yielded PD = 20.9 M0.19 (r = 0.96, P < 0.001). Analysis of published short circuit current (SCC, in microA/cm2) data revealed a similar relationship: SCC = 38.2 M0.21. Thus, the transepithelial electrical potential and active charge transport by the tracheal epithelium are allometric variables that may have direct physiological significance. These results raise questions regarding the importance of net osmotic solute and water transport across the tracheal epithelium.

Modulation of bronchial epithelial cell barrier function by in vitro ozone exposure.

The epithelial cells lining the small, peripheral airways function as important targets for the action of inspired ozone. Loss of epithelial barrier integrity in these regions is a common element in ozone-induced airway inflammation. To investigate the direct effect of ozone on epithelial barrier function, canine bronchial epithelial (CBE) cells grown with an air interface were exposed for 3 hr to 0.2, 0.5, or 0.8 ppm ozone or to air. Mannitol flux, used as an index of paracellular permeability, increased above air controls by 461%, 774%, and 1172% at the three ozone concentrations, respectively. Transcellular electrical resistance exhibited a dose-related decrease. The immediate effect of 0.8 ppm ozone on permeability was significantly inhibited by preincubation for 48 hr in the presence of 1 ng/ml vitamin E (33%) or 1 microM vitamin A (34%). Responses to 0.5 ppm or 0.8 ppm were inhibited by pretreatment of the cells with 0.1 microM of the actin polymerizing agent phalloidin (34% and 25% inhibition, respectively). The increases in permeability induced by 0.2 and 0.5 ppm ozone were attenuated by 54% and 22%, respectively, at 18 hr after exposure, whereas that to 0.8 ppm was further enhanced by 42% at this time. The effects of ozone are modulated by the availability of antioxidants to the cells and appear to be associated with cytoskeletal dysfunction in CBE cells. The data are consistent with a loss of barrier function linked to a direct oxidative effect of ozone on individual CBE cells and indicate that the reversible or progressive nature of this effect is dose dependent.

Effects of acute ozone exposure on the electrophysiological properties of guinea pig trachea.

Acute ozone (O3) exposures produce an increase in the apparent permeability of the tracheal epithelium, but the mechanism of this response is poorly understood. Comparison of previous studies suggests that qualitative differences may exist between measurements made in vivo or in vitro. To test this possibility we used both in vitro and in vivo electrophysiological techniques to investigate the effects of O3 exposure on guinea pig tracheal epithelium. Male Hartley guinea pigs were exposed to either 1 or 2 ppm O3 or to filtered air for 3 h and were studied 0, 6, or 24 h after exposure. Air-exposed animals had in vitro mean tracheal potential (VT) -32.0 +/- 1.5 mV, conductance (GTL) 2.18 +/- 0.22 mS/cm, short-circuit current (ISCL) 62.6 +/- 3.7 microA/cm, and diameter (D) 2.44 +/- 0.10 mm. In vitro properties after 1 ppm O3 exposure did not differ at any time point from control. Two parts per million O3 increased ISCL, but only at 6 h postexposure. The effect of O3 on ISCL was abolished by amiloride. There were no significant changes in VT, GTL, or D. In vivo tracheal potential under pentobarbital anesthesia was -19.7 +/- 1.7 mV. At 6 h postexposure to 2 ppm O3, but not at 0 or 24 h, in vivo VT was increased. Thus, acute exposure of guinea pigs to a high concentration of O3 caused a delayed increase in Na+ absorption by the trachea with no change in conductance. This indicates that paracellular permeability of guinea pig tracheal epithelium was not substantially increased by acute O3 and suggests that enhanced macromolecular uptake in this species probably occurs transcellularly. Furthermore, the increase of in vivo VT following O3 exposure is consistent with the in vitro response, indicating that in vivo/in vitro differences are not responsible for the discrepancies between previous electrophysiological and "permeability" studies.